Biosafety aspects of RNAi-based pests control.

While the overuse of classical chemical pesticides has had a detrimental impact on the environment and human health, the discovery of RNA interference (RNAi) offered the opportunity to develop new and sustainable approaches for pest management. RNAi is a naturally occurring regulation and defense mechanism that can be exploited to effectively protect crops by silencing key genes affecting the growth, development, behavior or fecundity of pests. However, as with all technologies, there is a range of potential risks and challenges associated with the application of RNAi, such as dsRNA stability, the potential for off-target effects, the safety of non-target organisms, and other application challenges. A better understanding of the molecular mechanisms involved in RNAi and in-depth discussion and analysis of these associated safety risks, is required to limit or mitigate potential adverse effects. © 2024 Society of Chemical Industry.

RNA-Based Control of Fungal Pathogens in Plants.

Our duty to conserve global natural ecosystems is increasingly in conflict with our need to feed an expanding population. The use of conventional pesticides not only damages the environment and vulnerable biodiversity but can also still fail to prevent crop losses of 20-40% due to pests and pathogens. There is a growing call for more ecologically sustainable pathogen control measures. RNA-based biopesticides offer an eco-friendly alternative to the use of conventional fungicides for crop protection. The genetic modification (GM) of crops remains controversial in many countries, though expression of transgenes inducing pathogen-specific RNA interference (RNAi) has been proven effective against many agronomically important fungal pathogens. The topical application of pathogen-specific RNAi-inducing sprays is a more responsive, GM-free approach to conventional RNAi transgene-based crop protection. The specific targeting of essential pathogen genes, the development of RNAi-nanoparticle carrier spray formulations, and the possible structural modifications to the RNA molecules themselves are crucial to the success of this novel technology. Here, we outline the current understanding of gene silencing pathways in plants and fungi and summarize the pioneering and recent work exploring RNA-based biopesticides for crop protection against fungal pathogens, with a focus on spray-induced gene silencing (SIGS). Further, we discuss factors that could affect the success of RNA-based control strategies, including RNA uptake, stability, amplification, and movement within and between the plant host and pathogen, as well as the cost and design of RNA pesticides.

Silencing a Chitinase Gene, PstChia1, Reduces Virulence of Puccinia striiformis f. sp. tritici.

Chitin is the main component of fungal cell walls, which can be recognized by pattern recognition receptors (PRRs) as pathogen-associated molecular patterns (PAMP). Chitinase in filamentous fungi has been reported to degrade immunogenic chitin oligomers, thereby preventing chitin-induced immune activation. In this study, we identified the chitinase families in 10 fungal genomes. A total of 131 chitinase genes were identified. Among the chitinase families, 16 chitinase genes from Puccinia striiformis f. sp. tritici (Pst) were identified, and the expression of PstChia1 was the highest during Pst infection. Further studies indicated that PstChia1 is highly induced during the early stages of the interaction of wheat and Pst and has chitinase enzyme activity. The silencing of PstChia1 revealed that PstChia1 limited the growth and reduced the virulence of Pst. The expression level of TaPR1 and TaPR2 was induced in PstChia1 knockdown plants, suggesting that PstChia1 is involved in regulating wheat resistance to Pst. Our data suggest that PstChia1 contributes to pathogenicity by interfering with plant immunity and regulating the growth of Pst.

Host-induced gene silencing of PcCesA3 and PcOSBP1 confers resistance to Phytophthora capsici in Nicotiana benthamiana through NbDCL3 and NbDCL4 processed small interfering RNAs.

Host-induced gene silencing (HIGS) is a RNA-based system depend on the biological macromolecules generated in plants to control diseases. However, the effector proteins active in the HIGS are uncertain, which impedes its further application, especially for oomycete that lack efficient HIGS targets. Phytophthora capsici is an important oomycete causes blight in over 70 crops. Here, we comprehensively screened efficient HIGS vectors targeting PcCesA3 or PcOSBP1 in P. capsici to better control it and explore the characteristics of efficient HIGS vectors. Among the 26 vectors with different lengths and structures, we found that hairpin vectors with a 70 nt loop and ~ 500 bp stem showed the highest control efficacy, with the expressing of the screened vectors, the infection and fertility of P. capsici were greatly inhibited in transgenic Nicotiana benthamiana. Based on these efficient vectors, we demonstrated that the amount of HIGS vector generated small interfering RNAs (siRNAs) was positively related to gene silencing efficiency and resistance, and that NbDCL3 and NbDCL4 were the key effectors producing siRNAs. This work discovers the principles for efficient HIGS vectors design, and elucidates the molecular mechanism of HIGS, which could benefit the control of many other plant diseases based on HIGS.

New Insights on the Integrated Management of Plant Diseases by RNA Strategies: Mycoviruses and RNA Interference.

RNA-based strategies for plant disease management offer an attractive alternative to agrochemicals that negatively impact human and ecosystem health and lead to pathogen resistance. There has been recent interest in using mycoviruses for fungal disease control after it was discovered that some cause hypovirulence in fungal pathogens, which refers to a decline in the ability of a pathogen to cause disease. Cryphonectria parasitica, the causal agent of chestnut blight, has set an ideal model of management through the release of hypovirulent strains. However, mycovirus-based management of plant diseases is still restricted by limited approaches to search for viruses causing hypovirulence and the lack of protocols allowing effective and systemic virus infection in pathogens. RNA interference (RNAi), the eukaryotic cell system that recognizes RNA sequences and specifically degrades them, represents a promising. RNA-based disease management method. The natural occurrence of cross-kingdom RNAi provides a basis for host-induced gene silencing, while the ability of most pathogens to uptake exogenous small RNAs enables the use of spray-induced gene silencing techniques. This review describes the mechanisms behind and the potential of two RNA-based strategies, mycoviruses and RNAi, for plant disease management. Successful applications are discussed, as well as the research gaps and limitations that remain to be addressed.

The master role of siRNAs in plant immunity.

Gene silencing mediated by small noncoding RNAs (sRNAs) is a fundamental gene regulation mechanism in eukaryotes that broadly governs cellular processes. It has been established that sRNAs are critical regulators of plant growth, development, and antiviral defence, while accumulating studies support positive roles of sRNAs in plant defence against bacteria and eukaryotic pathogens such as fungi and oomycetes. Emerging evidence suggests that plant sRNAs move between species and function as antimicrobial agents against nonviral parasites. Multiple plant pathosystems have been shown to involve a similar exchange of small RNAs between species. Recent analysis about extracellular sRNAs shed light on the understanding of the selection and transportation of sRNAs moving from plant to parasites. In this review, we summarize current advances regarding the function and regulatory mechanism of plant endogenous small interfering RNAs (siRNAs) in mediating plant defence against pathogen intruders including viruses, bacteria, fungi, oomycetes, and parasitic plants. Beyond that, we propose potential mechanisms behind the sorting of sRNAs moving between species and the idea that engineering siRNA-producing loci could be a useful strategy to improve disease resistance of crops.

RNA Interference: Promising Approach to Combat Plant Viruses.

Plant viruses are devastating plant pathogens that severely affect crop yield and quality. Plants have developed multiple lines of defense systems to combat viral infection. Gene silencing/RNA interference is the key defense system in plants that inhibits the virulence and multiplication of pathogens. The general mechanism of RNAi involves (i) the transcription and cleavage of dsRNA into small RNA molecules, such as microRNA (miRNA), or small interfering RNA (siRNA), (ii) the loading of siRNA/miRNA into an RNA Induced Silencing Complex (RISC), (iii) complementary base pairing between siRNA/miRNA with a targeted gene, and (iv) the cleavage or repression of a target gene with an Argonaute (AGO) protein. This natural RNAi pathway could introduce transgenes targeting various viral genes to induce gene silencing. Different RNAi pathways are reported for the artificial silencing of viral genes. These include Host-Induced Gene Silencing (HIGS), Virus-Induced Gene Silencing (VIGS), and Spray-Induced Gene Silencing (SIGS). There are significant limitations in HIGS and VIGS technology, such as lengthy and time-consuming processes, off-target effects, and public concerns regarding genetically modified (GM) transgenic plants. Here, we provide in-depth knowledge regarding SIGS, which efficiently provides RNAi resistance development against targeted genes without the need for GM transgenic plants. We give an overview of the defense system of plants against viral infection, including a detailed mechanism of RNAi, small RNA molecules and their types, and various kinds of RNAi pathways. This review will describe how RNA interference provides the antiviral defense, recent improvements, and their limitations.

Host-Induced Gene Silencing of a G Protein α Subunit Gene CsGpa1 Involved in Pathogen Appressoria Formation and Virulence Improves Tobacco Resistance to Ciboria shiraiana.

Hypertrophy sorosis scleroteniosis caused by Ciboria shiraiana is the most devastating disease of mulberry fruit. However, few mulberry lines show any resistance to C. shiraiana. An increasing amount of research has shown that host-induced gene silencing (HIGS) is an effective strategy for enhancing plant tolerance to pathogens by silencing genes required for their pathogenicity. In this study, two G protein α subunit genes, CsGPA1 and CsGPA2, were identified from C. shiraiana. Silencing CsGPA1 and CsGPA2 had no effect on hyphal growth but reduced the number of sclerotia and increased the single sclerotium weight. Moreover, silencing CsGpa1 resulted in increased fungal resistance to osmotic and oxidative stresses. Compared with wild-type and empty vector strains, the number of appressoria was clearly lower in CsGPA1-silenced strains. Importantly, infection assays revealed that the virulence of CsGPA1-silenced strains was significantly reduced, which was accompanied by formation of fewer appressoria and decreased expression of several cAMP/PKA- or mitogen-activated protein-kinase-related genes. Additionally, transgenic Nicotiana benthamiana expressing double-stranded RNA targeted to CsGpa1 through the HIGS method significantly improved resistance to C. shiraiana. Our results indicate that CsGpa1 is an important regulator in appressoria formation and the pathogenicity of C. shiraiana. CsGpa1 is an efficient target to improve tolerance to C. shiraiana using HIGS technology.

Optimizing RNAi-Target by Nicotiana benthamiana-Soybean Mosaic Virus System Drives Broad Resistance to Soybean Mosaic Virus in Soybean.

Soybean mosaic virus (SMV) is a prevalent pathogen of soybean (Glycine max). Pyramiding multiple SMV-resistance genes into one individual is tedious and difficult, and even if successful, the obtained multiple resistance might be broken by pathogen mutation, while targeting viral genome via host-induced gene silencing (HIGS) has potential to explore broad-spectrum resistance (BSR) to SMV. We identified five conserved target fragments (CTFs) from S1 to S5 using multiple sequence alignment of 30 SMV genome sequences and assembled the corresponding target-inverted-repeat constructs (TIRs) from S1-TIR to S5-TIR. Since the inefficiency of soybean genetic transformation hinders the function verification of batch TIRs in SMV-resistance, the Nicotiana benthamiana-chimeric-SMV and N. benthamiana-pSMV-GUS pathosystems combined with Agrobacterium-mediated transient expression assays were invented and used to test the efficacy of these TIRs. From that, S1-TIR assembled from 462 bp CTF-S1 with 92% conservation rate performed its best on inhibiting SMV multiplication. Accordingly, S1-TIR was transformed into SMV-susceptible soybean NN1138-2, the resistant-healthy transgenic T1-plants were then picked out via detached-leaf inoculation assay with the stock-plants continued for progeny reproduction (T1 dual-utilization). All the four T3 transgenic progenies showed immunity to all the inoculated 11 SMV strains under individual or mixed inoculation, achieving a strong BSR. Thus, optimizing target for HIGS via transient N. benthamiana-chimeric-SMV and N. benthamiana-pSMV-GUS assays is crucial to drive robust resistance to SMV in soybean and the transgenic S1-TIR-lines will be a potential breeding source for SMV control in field.

An Arabidopsis downy mildew non-RxLR effector suppresses induced plant cell death to promote biotroph infection.

Our understanding of obligate biotrophic pathogens is limited by lack of knowledge concerning the molecular function of virulence factors. We established Arabidopsis host-induced gene silencing (HIGS) to explore gene functions of Hyaloperonospora arabidopsidis, including CYSTEINE-RICH PROTEIN (HaCR)1, a potential secreted effector gene of this obligate biotrophic pathogen. HaCR1 HIGS resulted in H. arabidopsidis-induced local plant cell death and reduced pathogen reproduction. We functionally characterized HaCR1 by ectopic expression in Nicotiana benthamiana. HaCR1 was capable of inhibiting effector-triggered plant cell death. Consistent with this, HaCR1 expression in N. benthamiana led to stronger disease symptoms caused by the hemibiotrophic oomycete pathogen Phytophthora capsici, but reduced disease symptoms caused by the necrotrophic fungal pathogen Botrytis cinerea. Expressing HaCR1 in transgenic Arabidopsis confirmed higher susceptibility to H. arabidopsidis and to the bacterial hemibiotrophic pathogen Pseudomonas syringae. Increased H. arabidopsidis infection was in accordance with reduced PATHOGENESIS RELATED (PR)1 induction. Expression of full-length HaCR1 was required for its function, which was lost if the signal peptide was deleted, suggesting its site of action in the plant apoplast. This study provides phytopathological and molecular evidence for the importance of this widespread, but largely unexplored class of non-RxLR effectors in biotrophic oomycetes.

Host-induced silencing of the Colletotrichum gloeosporioides conidial morphology 1 gene (CgCOM1) confers resistance against Anthracnose disease in chilli and tomato.

KEY MESSAGE: Host mediated silencing of COM1 gene of Colletotrichum gloeosporioides disables appressorial differentiation and effectively prevents the development of Anthracnose disease in chilli and tomato. Anthracnose disease is caused by the ascomycetes fungal species Colletotrichum, which is responsible for heavy yield losses in chilli and tomato worldwide. Conventionally, harmful pesticides are used to contain anthracnose disease with limited success. In this study, we assessed the potential of Host-Induced Gene Silencing (HIGS) approach to target the Colletotrichum gloeosporioides COM1 (CgCOM1) developmental gene involved in the fungal conidial and appressorium formation, to restrict fungal infection in chilli and tomato fruits. For this study, we have developed stable transgenic lines of chilli and tomato expressing CgCOM1-RNAi construct employing Agrobacterium-mediated transformation. Transgenic plants were characterized by molecular and gene expression analyses. Production of specific CgCOM1 siRNA in transgenic chilli and tomato RNAi lines was confirmed by stem-loop RT-PCR. Fungal challenge assays on leaves and fruits showed that the transgenic lines were resistant to anthracnose disease-causing C. gloeosporioides in comparison to wild type and empty-vector control plants. RT-qPCR analyses in transgenic lines revealed extremely low abundance of CgCOM1 transcripts in the C. gloeosporioides infected tissues, indicating near complete silencing of CgCOM1 gene expression in the pathogen. Microscopic examination of the Cg-challenged leaves of chilli-CgCOM1i lines revealed highly suppressed conidial germination, germ tube development, appressoria formation and mycelial growth of C. gloeosporioides, resulting in reduced infection of plant tissues. These results demonstrated highly efficient use of HIGS in silencing the expression of essential fungal developmental genes to inhibit the growth of pathogenic fungi, thus providing a highly precise approach to arrest the spread of disease.

Double-Stranded RNA Technology to Control Insect Pests: Current Status and Challenges.

Exploiting the RNA interference (RNAi) gene mechanism to silence essential genes in pest insects, leading to toxic effects, has surfaced as a promising new control strategy in the past decade. While the first commercial RNAi-based products are currently coming to market, the application against a wide range of insect species is still hindered by a number of challenges. In this review, we discuss the current status of these RNAi-based products and the different delivery strategies by which insects can be targeted by the RNAi-triggering double-stranded RNA (dsRNA) molecules. Furthermore, this review also addresses a number of physiological and cellular barriers, which can lead to decreased RNAi efficacy in insects. Finally, novel non-transgenic delivery technologies, such as polymer or liposomic nanoparticles, peptide-based delivery vehicles and viral-like particles, are also discussed, as these could overcome these barriers and lead to effective RNAi-based pest control.

Host-Induced Gene Silencing of an Adenylate Kinase Gene Involved in Fungal Energy Metabolism Improves Plant Resistance to Verticillium dahliae.

Verticillium wilt, caused by the ascomycete fungus Verticillium dahliae (Vd), is a devastating disease of numerous plant species. However, the pathogenicity/virulence-related genes in this fungus, which may be potential targets for improving plant resistance, remain poorly elucidated. For the study of these genes in Vd, we used a well-established host-induced gene silencing (HIGS) approach and identified 16 candidate genes, including a putative adenylate kinase gene (VdAK). Transiently VdAK-silenced plants developed milder wilt symptoms than control plants did. VdAK-knockout mutants were more sensitive to abiotic stresses and had reduced germination and virulence on host plants. Transgenic Nicotiana benthamiana and Arabidopsis thaliana plants that overexpressed VdAK dsRNAs had improved Vd resistance than the wild-type. RT-qPCR results showed that VdAK was also crucial for energy metabolism. Importantly, in an analysis of total small RNAs from Vd strains isolated from the transgenic plants, a small interfering RNA (siRNA) targeting VdAK was identified in transgenic N. benthamiana. Our results demonstrate that HIGS is a promising strategy for efficiently screening pathogenicity/virulence-related genes of Vd and that VdAK is a potential target to control this fungus.

Host-Induced Silencing of Fusarium graminearum Genes Enhances the Resistance of Brachypodium distachyon to Fusarium Head Blight.

Fusarium head blight (FHB) caused by Fusarium pathogens are devastating diseases worldwide. Host-induced gene silencing (HIGS) which involves host expression of double-stranded RNA (dsRNA)-generating constructs directed against genes in the pathogen has been a potential strategy for the ecological sound control of FHB. In this study, we constructed transgenic Brachypodium distachyon lines carrying RNA interference (RNAi) cassettes to target two essential protein kinase genes Fg00677 and Fg08731, and cytochrome P450 lanosterol C14-α-demethylase (CYP51) encoding genes (CYP51A, CYP51B, and CYP51C) of Fusarium graminearum, respectively. Northern blotting confirmed the presence of short interfering RNAs (siRNA) derived from Fg00677, Fg08731, and CYP51 in transgenic B. distachyon plants, and the transcript levels of the corresponding genes were down-regulated in the F. graminearum colonizing B. distachyon spikes. All the corresponding independent, Fg00677-RNAi, Fg08731-RNAi, and CYP51-RNAi transgenic T2 lines exhibited strong resistance to F. graminearum, suggesting that silencing molecules produced by transgenic plants inhibited the corresponding gene function by down-regulating its expression, thereby reducing pathogenicity. Our results indicate that Fg00677 and Fg08731 are effective targets for HIGS and can be applied to construct transgenic HIGS materials to enhance FHB resistance in wheat and other cereal crops.

Small RNA Functions as a Trafficking Effector in Plant Immunity.

Small RNAs represent a class of small but powerful agents that regulate development and abiotic and biotic stress responses during plant adaptation to a constantly challenging environment. Previous findings have revealed the important roles of small RNAs in diverse cellular processes. The recent discovery of bidirectional trafficking of small RNAs between different kingdoms has raised many interesting questions. The subsequent demonstration of exosome-mediated small RNA export provided a possible tool for further investigating how plants use small RNAs as a weapon during the arms race between plant hosts and pathogens. This review will focus on discussing the roles of small RNAs in plant immunity in terms of three aspects: the biogenesis of extracellular small RNAs and the transportation and trafficking small RNA-mediated gene silencing in pathogens.

RNAi as an emerging approach to control Fusarium head blight disease and mycotoxin contamination in cereals.

Fusarium graminearum is a major fungal pathogen of cereals worldwide, causing seedling, stem base and floral diseases, including Fusarium head blight (FHB). In addition to yield and quality losses, FHB contaminates cereal grain with mycotoxins, including deoxynivalenol, which are harmful to human, animal and ecosystem health. Currently, FHB control is only partially effective due to several intractable problems. RNA interference (RNAi) is a natural mechanism that regulates gene expression. RNAi has been exploited in the development of new genomic tools that allow the targeted silencing of genes of interest in many eukaryotes. Host-induced gene silencing (HIGS) is a transgenic technology used to silence fungal genes in planta during attempted infection and thereby reduces disease levels. HIGS relies on the host plant's ability to produce mobile small interfering RNA molecules, generated from long double-stranded RNA, which are complementary to targeted fungal genes. These molecules are transferred from the plant to invading fungi via an uncharacterised mechanism, to cause gene silencing. Here, we describe recent advances in RNAi-mediated control of plant pathogenic fungi, highlighting the key advantages and disadvantages. We then discuss the developments and implications of combining HIGS with other methods of disease control. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

[Application of host induced gene silencing in crop protection against fungal diseases].

Fungal diseases are the main threat to crop yield and quality, often leading to huge economic losses. Chemical fungicides are almost useless to soil-borne and vascular fungal pathogens. The most effective way is crop resistance breeding by using resistance genes. Yet, for plants lacking resistance resources, new approaches are urgently needed for crop protection. Recently, host-induced gene silencing (HIGS) is developed based on the well-known RNA interference, and already effective against viruses and pests. However, it is challenging to validate HIGS in soil-borne fungal pathogens due to uncharacterized and complicated infection processes. Recently, we have made great progresses in revealing the infection structure of Verticillium dahliae, a soil-borne and vascular fungal pathogen that leads to verticillium wilt disease to many crops, including cotton plants. Moreover, we demonstrate that cotton exports endogenous microRNAs to inhibit virulence gene expression in V. dahliae. The most exciting achievement is the successful application of HIGS in cotton plants that confer resistance to V. dahliae. All these results reveal a promising potential for applying HIGS against a wide range of soil-borne fungi.

A unique invertase is important for sugar absorption of an obligate biotrophic pathogen during infection.

An increased invertase activity in infected plant tissue has been observed in many plant-pathogen interactions. However, the origin of this increased invertase activity (plant and/or pathogen) is still under debate. In addition, the role of pathogen invertases in the infection process is also unclear. We identified and cloned a gene with homology to invertases from Puccinia striiformis f. sp. tritici (Pst). Transcript levels of PsINV were analyzed by quantitative reverse transcription PCR in both compatible and incompatible Pst-wheat interactions . Function of the gene product was confirmed by heterologous expression, and its function in Pst infection was analyzed by host-induced gene silencing (HIGS). Pst abundantly secretes invertase during its invasion attempts whether in a compatible or incompatible interaction with wheat. Further research into the different domains of this protein indicated that the rust-specific sequence contributes to a higher efficiency of sucrose hydrolysis. With PsINV silenced by HIGS during the infection process, growth of Pst is inhibited and conidial fructification incomplete. Finally, pathogenicity of Pst is impaired and spore yield significantly reduced. Our results clearly demonstrate that this Pst invertase plays a pivotal role in this plant-pathogen interaction probably by boosting sucrose hydrolysis to secure the pathogen's sugar absorption.

Puccinia striiformis f. sp. tritici microRNA-like RNA 1 (Pst-milR1), an important pathogenicity factor of Pst, impairs wheat resistance to Pst by suppressing the wheat pathogenesis-related 2 gene.

Small RNAs (sRNAs), an important type of pathogenicity factor, contribute to impairing host immune responses. However, little is known about sRNAs in Puccinia striiformis f. sp. tritici (Pst), one of the most destructive pathogens of wheat (Triticum aestivum L.). Here, we report a novel microRNA-like RNA (milRNA) from Pst termed microRNA-like RNA 1 (Pst-milR1), which suppresses wheat defenses during wheat-Pst interactions. We identified Pst-milR1 as a novel milRNA in Pst. Biological prediction and co-transformation showed that Pst-milR1 takes part in cross-kingdom RNA interference (RNAi) events by binding the wheat pathogenesis-related 2 (PR2) gene. Silencing of the Pst-milR1 precursor resulted in increased wheat resistance to the virulent Pst isolate CYR31. PR2 knock-down plants increased the susceptibility of wheat to the avirulent Pst isolate CYR23. This suggests that Pst-milR1 represses the plant immune response by suppressing the expression of PR2. Taking our findings together, we postulate that Pst-milR1 is an important pathogenicity factor in Pst, which acts as an effector to suppress host immunity. Our results provide significant new insights into the pathogenicity of the stripe rust pathogen.